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From 2012.igem.org

And Introduction to Promoters

Promoters are upstream sequences that regulate transcription. Promoters are usually short sequences and act as binding sites for a variety of different RNA polymerases. Promoters have different binding affinities based on their sequence and can be characterized in a matter of different ways. Our project looks to measure some of these properties using fluorescence measurements. In our case, we are characterizing promoters that bind to RNA polymerase from the T7 phage. The T7 RNA polymerase binds to its promoter very tightly and produces a high amount of expression. The lac operator is a short sequence that binds to the LacI repressor, which prevents transcription. The LacI protein responds to lactose in the cell. Lactose analogs have been made which are not consumed by E. coli and "turn on" the gene of interest. Our promoters have different affinities to the T7 RNA polymerase and the LacI repressor and therefore have different measurable properties.

What is fluorescence, exactly?

Fluorescence is a property of some molecules, particularly aromatic organic dyes that absorb photons at a certain wavelength and emit them at a longer, lower energy wavelength.

Fluorescence is described using quantum mechanics principles and organic chemistry. Five and six-member rings tend to fluoresce brightly because of electron delocalization and the properties that are associated with electron delocalization through the p-orbitals. Fluorescent molecules come in a variety of flavors and uses based on their properties, and shape. Fluorescent molecules are known as fluorophores and can take the form of organic dyes or proteins. So far, many different types of fluorophores have been discovered, developed and studied in great detail. Typically, fluorescent proteins have a fluorophore that consists of a few side chains that react and form a complex similar to that of an organic dye. For example, GFP (the most common fluorescent protein) has an HBI fluorophore. Our Spinach construct binds to a dye that derives from this fluorophore. Fluorescence of a molecule can depend on conformation, in the case of our fluorogens, malachite green and DFHBI, which are conditional fluorophores, the molecule must be in a certain conformation to fluoresce. Otherwise, it will absorb photons, but it will emit them very inefficiently (extremely low quantum yield). Fluorescence is a widely studied phenomena and a lot of research is involved with improving current fluorescence technologies and its applications.

What is Spinach?

Spinach is an RNA sequence that can be expressed in cells (in this case, E. coli ) and fluoresces green when DFHBI (an organic dye) is bound to it that can be used to quantify RNA concentration in a cell. Spinach binds to an organic dye called DFHBI which doesn't fluoresce by itself but fluoresces very brightly when it is bound to Spinach. DFHBI is chemically derived from the chromophore in GFP but is altered to increase brightness when bound to RNA. Other fluorescent RNAs have been described but many are non-specific and have many unwanted functions like cytotoxicity. Other fluorescent RNAs also are difficult to quantify because the cells' RNAses (RNA destroying enzymes) can cut out the fluorescent sequence at unpredictable times, making quantification impractical. Spinach utilizes a scaffold that derives from a tRNA sequence, which disguises the RNA so that RNAses leave it alone. Manipulations to the sequence that Spinach is attached to allows for a variety of analyses functions. RNA can be arranged to bind to just about any small molecule in the same way that Spinach was developed (using a SELEX method) to track cellular metabolites. This allows for quantification of another important system in cells. However, in our system, Spinach is incorporated in the mRNA (between the promoter and the RBS) so mRNA is quantified. Click for more information on Spinach. Spinach is the first published RNA sequence of its kind and more sequence/dye combinations are in development; as a result, in years to come, multiple genes (both RNA and protein) can be analyzed in great detail simultaneously.

What is a FAP?

A fluorogen activating protein is a small (26-35kD) protein that derives from the variable region in an antibody. FAPs are not fluorescent unless a fluorogen (also not normally fluorescent) is added, in which case the FAP changes the conformation of the fluorogen and the complex fluoresces brightly. FAPs are currently used to tag certain proteins like actin or tubulin in mammalian cells. FAPs are not primarily expressed in E. coli although we have expressed certain FAPs in E. coli. The two main dyes that the current series of FAPs bind to are malachite green and thiazole orange; our construct uses a variant that binds to malachite green. These dyes are normally cell impermeable but can be designed to penetrate cell membranes. As a result, they were originally used to tag surface proteins. FAPs are excellent reporters because they are small proteins that are soluble and have virtually no maturation time and are highly photostable unlike traditional variants of GFP. FAP technology is widely unexplored but shows promise for new fluorescent technology. FAPs have been used to track individual molecules to 5nm definition as opposed to the typical 200nm. Engineered dyes, called dyedrons, have been developed that increase fluorescence intensity and can allow researchers to improve on live cell imaging techniques. FAPs are genetically different and respond to different excitation wavelengths so researchers can image multiple proteins at the same time in order to understand complex biological processes.

Why is this project important?

• The ability to monitor protein production with fluorescence is a growing field that promises advances in drug development and improving quality control in drug manufacturing.

• Promoter strength directly affects a cell's ability to perform typical functions like divide or move. Designing a genetic circuit that will not overload the cells is key in synthetic biology.

• Inducible promoters are widely used in synthetic biology but many are under-characterized.




Primary Objective: A New Set of Well-Characterized Promoters

Our primary objective is to develop a series of BioBricks that are well characterized based on our methods of measurement.

We assert that our new method of analyzing promoters can quantify certain properties such as:

1. Translational efficiency in vivo
2. in vivo transcription rates
3. Promoter strength
4. In vivo mRNA and protein half-lives in real time
The promoters we submit will be characterized with these properties.